Interleukin-1β and interleukin-6 disturb the antioxidant enzyme system in bovine chondrocytes: a possible explanation for oxidative stress generation  M. Mathy-Hartert, Ph.D., L. Hogge, B.Sc., C. Sanchez, Ph.D., G. Deby-Dupont, Ph.D., J.M. Crielaard, M.D., Y. Henrotin, Ph.D.  Osteoarthritis and Cartilage  Volume 16, Issue 7, Pages 756-763 (July 2008) DOI: 10.1016/j.joca.2007.10.009 Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Effect of IL-1β and IL-6 on SOD activity. A&C: Chondrocytes were incubated for 24h with or without increased concentrations of IL-1β or IL-6. B: Chondrocytes were incubated for 4–96h without or with IL-1β (0.18ng/ml) or IL-6 (100ng/ml). D: SOD activity assay was performed with KCN (1mM) to discriminate Mn and Cu/Zn SOD activities. Each condition was tested in triplicate. Results are expressed as mean±s.e.m. of three independent experiments. ***P<0.001 vs not stimulated with IL-1β; ○○○P<0.001 vs not stimulated with IL-6. Osteoarthritis and Cartilage 2008 16, 756-763DOI: (10.1016/j.joca.2007.10.009) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Effect of IL-1β on Mn (A), Cu/Zn (B) and EC (C) SOD gene expression. Chondrocytes were cultured for 24h with increasing amounts of IL-1β. Each condition was tested in triplicate. Experiments were performed three times and data from a representative experiment are shown. *P<0.05 or **P<0.01 or ***P<0.001 vs not stimulated with IL-1β. Osteoarthritis and Cartilage 2008 16, 756-763DOI: (10.1016/j.joca.2007.10.009) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Effect of IL-6 on Mn SOD gene expression. Chondrocytes were cultured for 24h with increasing amounts of IL-6. Each condition was tested in triplicate. Experiments were performed three times and data from a representative experiment are shown. ***P<0.001 vs not stimulated with IL-6. Osteoarthritis and Cartilage 2008 16, 756-763DOI: (10.1016/j.joca.2007.10.009) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Effect of IL-1β and IL-6 on GPX activity (A) and gene expression (B). A: Chondrocytes were incubated for 4–96h without or with IL-1β (0.18ng/ml). Each condition was tested in triplicate. Results are expressed as mean±s.e.m. of three independent experiments. B: Chondrocytes were incubated for 24h without or with increased amounts of IL-1β or IL-6. Each condition was tested in triplicate. Experiments were performed three times and data from a representative experiment are shown. **P<0.01 or ***P<0.001 vs not stimulated with IL-1β; ○○○P<0.001 vs not stimulated with IL-6. Osteoarthritis and Cartilage 2008 16, 756-763DOI: (10.1016/j.joca.2007.10.009) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Effect of IL-1β on CAT activity (A&B) and gene expression (C). A: Chondrocytes were incubated for 96h without or with increased amounts of IL-1β. Each condition was tested in triplicate. Results are expressed as mean±s.e.m. of three independent experiments. B: Chondrocytes were incubated for 4–96h without or with IL-1β (0.18ng/ml). Each condition was tested in triplicate. Results are expressed as mean±s.e.m. of three independent experiments. C: Chondrocytes were incubated for 24h without or with increased amounts of IL-1β or IL-6. Each condition was tested in triplicate. Experiments were performed three times and data from a representative experiment are shown. *P<0.05 or ***P<0.001: IL-1β treated groups vs control; ○○○P<0.001: IL-6 treated groups vs control. Osteoarthritis and Cartilage 2008 16, 756-763DOI: (10.1016/j.joca.2007.10.009) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Effects of NF-κB inhibitors on SOD activation by IL-1β. After 1h of preincubation with BAY11-7082 (black bars) or MG132 (gray bars) at indicated concentration, chondrocytes were treated with IL-1β (0.18ng/ml) for the next 24h. Cellular extracts were analyzed for SOD activity (A) or Mn SOD gene expression (B). Each condition was tested in triplicate. Results are expressed as mean±s.e.m. of three independent experiments. ***P<0.001: drug treated groups were significantly different from the IL-1β treated groups. Osteoarthritis and Cartilage 2008 16, 756-763DOI: (10.1016/j.joca.2007.10.009) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 Effects of MAPK (black bars) and NF-κB inhibitors (gray bars) on GPX gene expression. After 1h of preincubation with SB203580 (20μM), SP600125 (10μM), PD98059 (10μM), BAY11-7082 (10μM) or MG132 (5μM), chondrocytes were treated with IL-1β (0.18ng/ml) for the next 24h. Cellular extracts were analyzed for GPX gene expression. Experiments were performed three times and data from a representative experiment are shown. **P<0.01 or ***P<0.001: drug treated groups were significantly different from the IL-1β treated group. Osteoarthritis and Cartilage 2008 16, 756-763DOI: (10.1016/j.joca.2007.10.009) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions